A Critical Role of Platelets and the Par4 Receptor in Developmental Growth Arrest of Thrombomodulin Null Embryos.

The transmembrane thrombin receptor, thrombomodulin (TM) suppresses excessive activation of blood coagulation. Complete deficiency of TM results in developmental growth arrest and embryonic lethality in mice. These embryos are resorbed by 8.5 day of development, and do not show “head turning” characteristic of the next developmental stage. The mechanism underlying this phenomenon has remained enigmatic. Growth arrest of TM null embryos is mediated by lack of TM expression in fetal-origin cells of the placenta, but is not associated with increased fibrin clots. TM null embryos cannot be rescued by anticoagulation therapy of the mother with heparin or by absence of maternal and fetal fibrinogen. Lethality of TM null embryos is, however, prevented by genetic reduction of embryonic tissue factor expression, suggesting the involvement of coagulation activation in its pathophysiology. We investigated into the role of platelets in this developmental defect. Using appropriate genetic crosses we generated TM null embryos in NF-E2 −/− mice and examined their fate during gestation. Deficiency of NF-E2 has been previously shown to be associated with severe reduction in platelet counts. We show that TM null embryos are rescued from developmental growth arrest in NF-E2 null mothers. We next wanted to determine whether rescue is specific to the absence of maternal platelets or to the absence of the transcription factor NF-E2. We injected pregnant mice from TM +/− intercrosses with platelet depleting antibodies, during midgestation. Antibody induced depletion of maternal platelets also restored the development of TM null embryos. These results demonstrate that in the absence of TM expression in fetal origin cells of the placenta, maternal platelets disrupt placental function resulting in developmental growth arrest of the embryo. To determine if thrombin-mediated platelet activation is required for the deleterious effect of maternal platelets on TM null embryos, we examined the fate of TM null embryos in Protease Activated Receptor 4 (Par4) null mice. Par4 signaling is essential for the activation of mouse platelets by thrombin. Our results show that the absence of maternal Par4 does not rescue TM null embryos. However, anticoagulation therapy of the mother with enoxaparin, in combination with the genetic absence of maternal Par4, results in “turned” emobryos past the developmental block of untreated embryos. Enoxaparin treatment alone is not sufficient to rescue the embryos. These observations implicate maternal platelets, thrombin and the Par4 receptor in mediating the developmental growth arrest of TM null embryos. Toxic effects of platelet mediated excessive thrombin generation, or chemokine secretion, induced by activation of the platelet Par4 receptor are candidate mechanisms. We have substantiated our observations on the role of maternal platelets in pregnancy outcome in another mouse model of fetal loss that we have generated. In this model, combining maternal thrombophilia due to Factor V Leiden polymorphism with embryonic TM Pro mutation results in fetal loss. Depleting platelets in maternal peripheral blood by antibody treatment of pregnant mice rescues the embryos from midgestational lethality. Enoxaparin treatment, on the other hand, is ineffective. Our results highlight an important role of maternal platelets in determining pregnancy outcome. Understanding this role may open avenues for prevention and treatment of pregnancy disorders in prothrombotic mothers through attenuation of platelet function.